Spatial variability of the Po River food web and its comparison with the Danube River food web

Freshwater ecosystems are experiencing unprecedented pressure globally. To address environmental challenges, systematic and comparative studies on ecosystems are needed, though mostly lacking, especially for rivers. Here, we describe the food web of the Po River (as integrated from the white literature and monitoring data), describe the three river sections using network analysis, and compare our results with the previously compiled Danube River food web. The Po River food web was taxonomically aggregated in five consecutive steps (T1-T5) and it was also analyzed using the regular equivalence (REGE) algorithm to identify structurally similar nodes in the most aggregated T5 model. In total, the two river food webs shared 30 nodes. Two network metrics (normalized degree centrality [nDC]) and normalized betweenness centrality [nBC]) were compared using Mann-Whitney tests in the two rivers. On average, the Po River nodes have larger nDC values than in the Danube, meaning that neighboring connections are better mapped. Regarding nBC, there were no significant differences between the two rivers. Finally, based on both centrality indices, Carassius auratus is the most important node in the Po River food web, whereas phytoplankton and detritus are most important in the Danube River. Using network analysis and comparative methods, it is possible to draw attention to important trophic groups and knowledge gaps, which can guide future research. These simple models for the Po River food web can pave the way for more advanced models, supporting quantitative and predictive—as well as more functional—descriptions of ecosystems.


Introduction
Global environmental challenges call for systematic and comparative studies on ecosystems. This requires standard sampling methods, data management, and well-established methods providing results that are either testable or (at least) provide support for planning empirical work. Despite of large number of field campaigns and huge amount of primary data, it is still quite in infancy to perform fully comparable analyses on several ecosystems, even if of similar Based on environmental homogeneity and aquatic community composition, we identified three sections in the Po River (Fig 1). The Upper Po River section from the source to Turin city (125 km long) is characterized by cold and turbulent waters, limited water flow, steep slope, predominantly boulder and pebble bed until the slope weakens and with it the turbulence and coarseness of the substrate, allowing colonization by rheophilic cyprinids. The Middle Po River section from Turin city to the Secchia River confluence (543 km long) is characterized by low slope, higher flow rate, general heating of the water, the connection to a system of lateral (off-channel) habitats, that are beginning to become complex and they assume an important role for the river ecosystem itself, and which in turn enhances species richness. Finally, the Lower Po River section, from the Secchia River confluence to the Po River Delta (127 km long), is characterized by deep, warmer and slower flow and less surface turbulence waters compared to other river sections, with the presence of euryhaline species. The high ecological, as well as socio-economic, value make the Po River a case study of European importance.

Data
Initially, a Web Of Science literature survey was conducted using the "Po River" term and the following keywords: "food web", "interaction", "prey", "predator, "network", "trophic", "feeding", "gut content", "isotope", "diet" (accesssed on April 28, 2022). This search found 279 articles. Studies on terrestrial species and interactions (e.g., Po floodplain) were not considered. The abstracts were screened, from which only 7 articles were found to be relevant. After reviewing these selected papers, two articles had data on rotifer predator-prey interactions [30,31]; two papers had detailed species list, but lacking diet information [32,33]; and three papers had information on plankton and macroinverterbrate community composition [34][35][36]. Therefore, it was not possible to obtain data for the Po River food web based only on white literature. Our research was complemented with survey data for fish, invertebrates, and plankton (see S1 Table for references). Information on predator-prey relationships was obtained from FishBase [37] and from published books [21,38]. Where available, size information (small/ large) and age information (juvenile/adult) were added to the database.

Network construction and analyses
Data was compiled for each river section (i.e., Upper, Middle, and Lower Po River sections) separately and merged into a master network (S2 Table). The network nodes (species or functional groups) are listed exactly as mentioned in the original sources. We decided to exclude the two articles specifically focused on the rotifera community [30,31], because they would have produced an isolated rotifera network (rotifers eating other rotifers). Although this subsystem is indeed very important, it realistically needs to be linked to the rest of the community, thus is something that needs further future consideration. After compiling all data, we followed the taxonomic aggregation procedure detailed in Patonai and Jordán (2021) for the Danube River [9], for comparability and standardization of methods. This includes five consecutive steps (Table 1) with a minor modification at step two (having detailed fish data, fish species were also aggregated into taxonomic families). In the first step (T1), the master dataset was aggregated so that the size (small/large) and age (juvenile/adult) information was combined at species level. In the second step (T2), we combined fish species into fish families (e.g., Silurus glanis into Siluridae). In the third step (T3), the two main fish orders were created (Cypriniformes, Perciformes). In step four (T4), invertebrates were aggregated into main groups (Annelida, Gastropoda, Mollusca, Crustacea, Insecta, Turbellaria). Finally, in step five (T5), all producers were aggregated, including nodes such as algae, diatoms, phytoplankton, and macrophytes. The detritus node remained separate (unaggregated). We note that the aggregation steps can be done in any order (since they represent separate taxonomic groups) and the effect of each aggregation step can be quantified using network analysis. We followed this order, because it logically goes from the smallest (species-level aggregation), through intermediate steps (taxonomic family, order, class), to the broadest category (producers).
Prior to analyses, binary (unweighted) networks were made symmetric (taking the interaction sums between two nodes, x ij + x ji ) in UCINET software [39]. Network visualization was done in R software version 4.2.0 [40] using Sankey plots in the 'networkD3' package [41]. The final aggregated (T5) river section networks were also clustered using the regular equivalence (REGE) algorithm, which groups nodes based on topological similarity [24,42], essentially quantifying trophic guilds based on food web structure [23].
Global network metrics. Global network metrics were computed in UCINET software [39]. Six global metrics were computed: number of nodes (N), number of links (L), network density (D), clustering coefficient (CL), average path length (d), and small-world index (SW). Number of nodes (N) is the number of species or functional groups in the network. Number of links (L) describes the number of connections between the nodes. Both of these measures give an indication of the complexity of the network. Network density (D) or connectance is Bythotrephes Carassius auratus Chydorus ovalis Chydorus sphaericus Cobitis bilineata x x x (Continued )  Master  T1  T2  T3  T4  T5 47 Cyclops vicinus Eudiaptomus gracilis Eudiaptomus padanus  Master  T1  T2  T3  T4  T5 94 Lepomis gibbosus Leuciscus aspius Micropterus salmoides Misgurnus anguillicaudatus  Master  T1  T2  T3  T4  T5 141 Moina micrura Phoxinus phoxinus Pleuroxus aduncus Pleuroxus denticulatus Rhodeus sericeus the number of actual links divided by the number of possible links [43]. It gives a quick snapshot of specialists vs generalists in the network (e.g., more generalists increase density, but more specialists decrease density). Network density is computed as D = 2L/N(N-1), where N (number of nodes) and L (number of links). Clustering coefficient (CL) is a measure of cohesion, it is the probability that two neighboring nodes n and m also share the neighbor node i. The weighted overall clustering coefficient reported is the"weighted mean of the clustering coefficient of all the nodes each one weighted by its degree" [39]. The average path length (d) is the mean distance between each node. In unweighted connectance webs, the distance is simply the number of links between two nodes. . Networks found in nature are found between the spectrum of random and regular graphs [44]. Local network metrics. Two local network centrality measures were computed. Normalized degree centrality (nDC) describes each node by the number of direct connections of a given node divided by N-1 [43]. It indicates how richly connected is a particular node considering its immediate neighborhood. Normalized betweenness centrality (nBC) for node i is the number of shortest paths between each pair of node j and k, containing node i [43]. It indicates a particular node's importance, in which large values suggest that the node acts as a bridge within the network structure. These two metrics were calculated in UCINET [39].

Comparison with the Danube River trophic network
The Danube is the second longest river in Europe, traversing over several countries from the Black Forest in Germany to the Black Sea in Romania [45]. Similarly to the Po River, it also has three main river sections [45] and a connectance web has been already compiled [9], which makes it possible to compare the two rivers. We were interested in what information is available for both rivers and what are the knowledge gaps. The two rivers were compared using the previously described local network metrics (nDC, nBC). Comparative and standardized methodology is crucial when comparing ecosystems.

Results and discussion
The global network metrics and the aggregation steps are summarized in Table 2 and visualized in Sankey plots (Fig 2). The global metrics give a snapshot of the consecutive networks. The Middle Po River had the most nodes and links, the Upper Po was comparable, and the Lower Po River had the fewest nodes and links (Table 2). Small fluctuations in the first steps were insignificant, whereas the changes in network metrics in the final aggregation steps (T4-T5) were more substantial. The T4 and T5 aggregation steps involved taxonomically diverse groups (invertebrates, producers), hence they had large effect on the networks. In each case, the number of nodes (N) and links (L) monotonically decreased through the aggregation process. The aggregation made the network nodes more homogenous. Network density (D) and clustering coefficient (CL) were generally variable in the first steps, but then increased in the last steps (T3-T5), making the final networks more dense and clustered. The average distance (d) decreased in all cases, except in the Upper Po, where it slightly increased at T2 and T3, but then decreased. Small-world index (SW) increased during the aggregation steps, ranging from i) very small values descriptive of random networks; to ii) larger numbers in the more aggregated versions, descriptive of other natural networks [44]. These global metrics are comparable in all three river sections and the master network, indicating that the most aggregated networks can be described as clustered (high density and clustering coefficients with reduced average network distances), and they are the most comparable to natural networks (i.e., intermediate SW values). The regular equivalence (REGE) algorithm examined these networks from a different perspective. Results of the REGE clustering for each river section are found in Fig 3. The REGE algorithm highlights the level or structural similarity the nodes (organisms) have in each river section. For example, Insecta and Crustacea nodes have the same functional role in the three PLOS ONE river sections. Hydracarina (water mites commonly found in freshwaters) are structurally grouped with detritus and producers (because all three nodes only have consumers eating them). This nicely shows that REGE highlights topological resemblance instead of the taxonomic approach. For the fish groups, REGE gave interesting insights. For some fish groups, the topological similarity reflected some difference from the taxonomically-derived Sankey plots. In the Upper Po River, Cypriniformes has closer resemblance to the invertebrate groups (probably due to being a predator as well as a prey), whereas the other five fish groups (i.e. Cottidae, Esocidae, Petromyzontidae, Perciformes, and Salmonidae) are grouped together into

PLOS ONE
one cluster (Figs 2a and 3a). In the Middle Po River, the two methods were similar for fish (Figs 2b and 3b) resulting in two fish clusters (higher trophic level fish: Esocidae, Salmonidae, Siluridae, Acipenseridae; and intermediate-and lower trophic-level fish: Anguillidae, Mugilidae, Perciformes, Cypriniformes, and Cyprinodontidae). In the Lower Po River, the two main REGE fish clusters (higher trophic level fish, mostly predators: Perciformes, Acipenseridae, Siluridae; and intermediate trophic-level fish: Anguillidae, Mugilidae, Cypriniformes, Fig 3c) nicely match the Sankey plot (Fig 2c). The self-loop in the Lower Po indicates cannibalism (Cypriniformes eating other Cypriniformes) (Fig 2c). Overall, the REGE clusters provide additional structural support for the Sankey plots, which are purely used for visualization purposes. However, the Sankey plots are informative due to the width of the fluxes (representing the number of nodes associated with each aggregated group). In the Upper Po River, the main diet of the larger aggregate fish groups (e.g., Salmonidae, Perciformes, Cypriniformes) are insects more than other invertebrates (e.g., Crustacea, Gastropoda, Annelida) (Fig 2a). In the Middle Po River, this is still true, but the importance of Crustacea in the diet increased for Cypriniformes, though not for Perciformes (Fig 2b). In the Lower Po River, the overall importance of Crustacea in the diet of various fish groups increased (Fig 2c).
In comparing the Po River with the Danube River, we found that they share 30 nodes (Table 3). On average, the Po River nodes have larger nDC values than in the Danube (median po = 0.110, median danube = 0.008, n = 30, Mann-Whitney U = 52.5, p<0.0001), meaning that neighboring connections are better mapped in the Po River. Interestingly, the Danube River also had lower nDC values when comparing invasive species in Lake Balaton (Hungary) [9], suggesting that the Danube River is the least well mapped (e.g., least amount of data) or that these species are ecologically not as well connected in the Danube River. Some nodes have comparable nDC values in both rivers (e.g., phytoplankton, Moina micrura, Sander lucioperca, Table 3, Fig 4a), suggesting that these taxa play a similar role in the trophic web of the Po and Danube Rivers. Regarding betweenness, there were no significant differences between the two rivers (median po = 0.571, median danube = 0.083, n = 30, Mann-Whitney U = 339, p = 0.102). The analysis showed that phytoplankton is important in both rivers (Table 3, Fig 4b), which is an evident result and can be extended to eutrophic rivers. Considering the nBC values of the shared species, some were more important in the Po River food web (Carassius auratus, Abramis brama, Rutilus rutilus), and others were more important in the Danube River food web (Chironomidae, Detritus, Perca fluviatilis). This means, that these nodes have a unique position within the network, frequently being on the shortest path between many nodes. Finally, based on both indices in the Po River, the nonnative Carassius auratus is most important, whereas phytoplankton has higher betweenness than degree, and the non-natives Abramis brama and Rutilus rutilus are more important considering nDC than for nBC (Fig 4c). In the Danube River, phytoplankton and detritus are most important for both indices, whereas Chironomidae has higher betweenness than degree, and Gymnocephalus cernua is more important considering nDC than for nBC (Fig  4d). Contrary to the Danube River, these results suggest that non-native species are important trophic players in the Po River, at the expense of native species, confirming that the Po River is facing an invasive species crisis [46][47][48][49]. These nodes have been pointed out by network analysis and should be further examined for their ecological importance in each system. Some of the differences might be real ecological differences between the two rivers (e.g., the importance of Carassius auratus is higher in the Po than in the Danube), but others might simply be from the fact that they are better mapped in the Po than in the Danube (e.g., Abramis brama). Using connectance webs, the interpretations are limited and should only be used to point out important groups to be investigated in the future or those that are missing information.

Conclusions
In this study, we aimed to highlight the importance of integrating monitoring data with network analysis for an ecosystem-level approach: the connectance web of the Po River and its main three river sections was compiled. This approach marks the need for a replicated approach for rivers considering their entire courses. We also emphasize the importance of using comparative methodology in order to be able to draw similarities and point out differences between different ecosystems. The Danube River food web was compiled using the same methodology, but highlighted different groups based on network analysis. Using aggregation, each network can be used for different purposes, but we recommend using the most aggregated versions for future modelling work (e.g., quantified networks, energy fluxes between the main compartments). We also found that the Sankey graphs (purely for visualization) and the REGE structural analysis nicely complement each other.